This revised competing renewal application of NIH RO1-CA111422: Cyclin D as a Retinoid Differentiation Molecular Target continues in depth studies of the important role of cyclin D1 as a major target of all-trans- retinoic acid (RA, retinoid) response by now examining how this is also a target of the hedgehog (Hh) pathway, also retinoid regulated, but in a previously unrecognized manner critical for differentiation and self- renewal of germ cell tumors (GCTs), the malignant counterparts of human embryonal stem (ES) cells. Intriguingly, our studies uncovered a unique retinoid response: induction of the Patched-1 (Ptch1) receptor which drives loss of self-renewal and regulates differentiation. Ptch1 is a target gene of the Hh pathway and is used as a read-out of Hh activation. The hypothesis explored is RA represses Hh signaling by a novel mechanism, Ptch1 induction, independent of smoothened (Smo), the only recognized target of Hh inhibitors, such as cyclopamine. Recognizing importance of this came from mining gene profiling studies successfully used by our team to identify unique retinoid targets in embryonal cancers (ECs). This proposal explores a distinct pharmacologic mechanism regulating self-renewal of ECs and likely other tumors. We are eager to pursue the Specific Aims that: (1) discover whether RA induction of Ptch1 is a general mechanism engaged by EC cells independent of Smo and if this is a common pathway of agents that trigger terminal differentiation of pluripotent EC cells; (2) probe the functional importance of this pathway by gain and loss of Ptch1 expression studies that affect Hh targets and regulate apoptosis, self-renewal or differentiation response; and (3) elucidate in vivo and clinical relevancy by learning whether RA induces Ptch1 in ES cells (or other cancer or stem cells) as well as in Ptch1 reporter mice and by confirming Ptch1 and its targets are differentially expressed in a unique tumor bank enabling comprehensive analyses of differentiated (mature teratoma) versus undifferentiated (EC) GCTs. This has successfully uncovered D-type cyclins as key regulators of human EC growth, chemotherapy response and differentiation. These unique in vitro, in vivo, and clinical resources with pharmacologic, cell and molecular biologic approaches will provide critical insights into the Hh pathway in regulating apoptosis, self-renewal or differentiation of GCT or other tumors. PUBLIC HEALTH RELEVANCE: This project studies how all-trans-retinoic acid, an FDA approved Vitamin A derivative, causes repression of the hedgehog pathway through a previously unrecognized mechanism that induces its receptor, Patched. This mechanism is active in embryonal cancers and stem cells. This project is relevant to embryonal cancer cells, the malignant counterpart of normal stem cells and successful conclusion of this project will provide insights into the retinoid role of targeting the hedgehog pathway in cancer to suppress self-renewal or growth and induce apoptosis or differentiation in these cells.